Identification of LMAN1- and SURF4-Dependent Secretory Cargoes

Abstract

Most proteins secreted into the extracellular space are first recruited from the endoplasmic reticulum into coat protein complex II (COPII)-coated vesicles or tubules that facilitate their transport to the Golgi apparatus. Although several secreted proteins have been shown to be actively recruited into COPII vesicles and tubules by the cargo receptors LMAN1 and SURF4, the full cargo repertoire of these receptors is unknown. We now report mass spectrometry analysis of conditioned media and cell lysates from HuH7 cells CRISPR targeted to inactivate the LMAN1 or SURF4 gene. We found that LMAN1 has limited clients in HuH7 cells, whereas SURF4 traffics a broad range of cargoes. Analysis of putative SURF4 cargoes suggests that cargo recognition is governed by complex mechanisms rather than interaction with a universal binding motif..

Keywords: COPII trafficking; cargo receptor; secretome.

Figure 1

Identifying proteins dependent on LMAN1 or SURF4 for efficient secretion. (A) Experimental design to identify LMAN1 and SURF4 cargoes in the human hepatoma cell line (HuH7). HuH7 cells were infected with lentiviruses delivering CRISPR/Cas9 and guide RNAs targeting LMAN1, SURF4, or a nontargeting (NT) control. Following selection, cells were cultured for 2 weeks before being switched to serum-free media for 12 h. Conditioned media and cell lysates were collected for protein identification and quantification by liquid chromatography (LC) followed by tandem mass tag (TMT) mass spectrometry (MS). A protein abundance ratio was calculated for each protein that was detected in both the media and lysate fractions as described in Methods. (B) Number of proteins with (red) and without (blue) a signal peptide identified in different M/L fractions. The number of signal peptide-containing proteins in each bar is indicated.

Figure 2

Differential effects on protein secretion in HuH7 cells following LMAN1 or SURF4 deletion. (A,B) Volcano plots comparing protein M/L ratios in LMAN1- (A) or SURF4- (B) deficient cells with those in controls. The log2 fold change (log2 fc) and statistical significance are plotted on the x- and y-axes, respectively. Proteins with a signal peptide are colored in red, and proteins without a signal peptide are colored in blue. Dashed vertical lines represent the log2 fc of 1 and −1. Dashed horizontal lines represent the −log10(q-value of 0.05). (C,D) Comparison of total abundance (estimation from FragPipe; see Methods) in the media with LMAN1 (C) or SURF4 (D) dependency. Trend lines represent linear regression. Dot sizes are proportional to −log10(q-value). R² values in blue and red are for proteins without and with an annotated signal peptide, respectively.

Figure 3

Most SURF4 cargoes do not contain an ER-ESCAPE motif. (A) Venn diagram of putative SURF4 cargoes that were identified in the current study, or previously reported by Gomez-Navarro et al.(24) in HEK293T and HuH7 cells following analyses of conditioned media, or by Huang et al.(36) using an in vitro COPII vesicle formation assay. (B) Classification system for the three amino acid residues immediate downstream of the signal peptide cleavage site (ER-ESCAPE tripeptide motif) proposed by Yin et al.(29) (C) Tripeptide motifs in previously reported SURF4 cargoes,^– in cargoes that were identified in more than one MS-based data sets, and in this study color-coded according to (B). (D) Percentage of each amino acid class [as described in (B)] at each position in the tripeptide motif for all secreted proteins with an annotated signal peptide in the proteome (n = 2110, see Methods) and for SURF4 candidate cargoes (n = 86). P-values were obtained from Fisher’s exact test.